Gene silencing

ABSTRACT

Methods are disclosed for screening of the occurrence of gene silencing (e.g. post transcriptional gene silencing) in an organism. Also provided are methods for isolating silencing agents so identified.

TECHNICAL FIELD

[0001] The present invention relates generally to methods and materialsfor use in achieving and detecting gene silencing, particularlypost-transcriptional gene silencing, in an organism.

PRIOR ART

[0002] Methods of detecting and efficiently achieving gene silencing areof great interest to those skilled in the art.

[0003] Post-transcriptional gene silencing (PTGS) is a nucleotidesequence-specific defence mechanism that can target both cellular andviral mRNAs. PTGS occurs in plants and fungi transformed with foreign orendogenous DNA and results in the reduced accumulation of RNA moleculeswith sequence similarity to the introduced nucleic acid (1, 2).

[0004] PTGS in plants can be suppressed by several virus-encodedproteins (6) and is closely related to RNA-mediated virus resistance andcross-protection in plants (7,8). Therefore, PTGS may represent anatural antiviral defence mechanism and transgenes might be targetedbecause they, or their RNA, are perceived as viruses. PTGS could alsorepresents a defence system against transposable elements and mayfunction in plant development (9-11). To account for the sequencespecificity, and post-transcriptional nature of PTGS it has beenproposed that antisense RNA forms a duplex with the target RNA therebypromoting its degradation or interfering with its translation (12).

[0005] One problem which exists in actually utilising efficient genesilencing, for instance via anti-sense mechanisms, is selectingappropriate regions to target. This problem has been reviewed in theliterature (see Szoka (1997) Nature Biotechnology 15: 509; Eckstein(1998) Nature Biotechnology 16: 24). Proposed solutions to selectinggood target regions include computational analysis (Patzel andSczakiel(1998) Nature Biotechnology 16: 64-68) or Rnase H cleavage usingchimeric anti-sense oligonucleotides (see Ho (1996) Nucleic Acid Res 24:1901-1907; Ho et al (1998) Nature Biotechnology 16: 59-62). Other groupshave used wide array of oligonucleotides to select those which formheteroduplexes (see Milner et al (1997) Nature Biotechnology 15:537-541).

DISCLOSURE OF THE INVENTION

[0006] The present inventors have investigated PTGS of target genesinitiated by a variety of silencing mechanisms in different organisms,and have established that in every case a previously uncharacterisedspecies of antisense RNA complementary to the targeted mRNA wasdetected. These RNA molecules were of a uniform length, estimated ataround 25 nucleotides, and their accumulation required either transgenesense transcription or RNA virus replication. Corresponding sense RNAmolecules were also detected.

[0007] There have been no previous reports of such short sense andantisense RNA molecules (hereinafter, collectively, SRMs) that aredetected exclusively in organisms exhibiting PTGS, possibly because(owing to their size) they could not have been readily detected byroutine RNA analyses.

[0008] It appears that the SRMs may be synthesized from an RNA templateand represent a specificity determinant and molecular marker of PTGS.Because of their correlation with PTGS and the nature of the molecules(short complementary molecules which could base pair with the targetRNAs) they are believed to represent a signal and/or inducer oractivator of PTGS.

[0009] The identification of this species by the present inventors maybe utilised by those skilled in the art in a variety of methods andprocesses which are discussed in more detail below. Generally speakingthe present invention provides, inter alia, methods of identifying andscreening for gene silencing and particular silenced genes in organisms;processes for producing or isolating silencing agents, and such isolatedagents themselves; methods for selecting target regions of nucleic acidswhich it is desired to silence and methods for silencing targetgenes-using the agents or target regions generated as above. Alsoincluded are relevant materials (e.g. nucleic acids, constructs, hostcells, transgenic plants, silenced organisms) and methods of use ofthese.

[0010] Importantly, the disclosure herein provides evidence that SRMsmay be a common mediator of PTGS in both plants and higher organisms,such as the nematode discussed in the Examples hereinafter. It waspreviously known that double stranded RNA induces a similar effect toplant PTGS in nematodes, insects (4) and protozoa (5). For instance PTGShas been demonstrated in Caenorhabditis elegans (a nematode worm) usingDsRNA introduced into the worms by microinjection, imbibing or byallowing the worms to eat bacteria (E. coli) which are synthesizingdsRNA. There was also some evidence that in some examples of PTGS inplants and dsRNA interference in nematodes, a signal is produced whichspreads and amplifies the silencing beyond the point of introduction ofthe original inducer of silencing. Although there were known to becertain similarities between the DsRNA induced silencing in nematodesand the causes of PTGS in plants, there was no clear evidence that thetwo are related.

[0011] Aspects of the invention will now be discussed in more detail.

[0012] Thus in one aspect of the present invention there is provided amethod of detecting, diagnosing, or screening for gene silencing in anorganism, which method comprises the steps of:

[0013] (i) obtaining sample material from the organism,

[0014] (ii) extracting nucleic acid material therefrom,

[0015] (iii) analysing the extracted nucleic acid in order to detect thepresence or absence of SRMs therein,

[0016] The result of the analysis in step (iii) may be correlated withthe presence of silencing in the organism.

[0017] The ‘sample’ may be all or part of the organism, but will includeat least some cellular material.

[0018] The term ‘SRMs’ is used to describe the short RNA moleculesdescribed herein which are approximately 25 nucleotides in length. Thesize appears to be very characteristic, being estimated as approximately25 nucleotides in all the cases tested (relative to the same molecularsize markers when assessed by chromatography). However, it may beslightly more or less than this characteristic length (say plus or minus1, 2, 3, 4 or 5 nucleotides) and where the term ‘25 nt RNA’ is usedherein, it will be understood by those skilled in the art that thecomments would apply equally in the event that the SRMs do not have thisprecise length.

[0019] Indeed the precise length may not be important, since thedisclosure herein permits the identification, isolation and utilisationof SRMS in any case.

[0020] In performing the invention, it may be preferred to analyse orotherwise utilise short anti-sense RNA molecules (SARMs) rather thanshort sense RNA molecules (SSRMs). Nonetheless, where reference is madeherein to SARMs (except where context clearly suggests otherwise) itwill be appreciated by those skilled in the art that the SSRMs may alsobe used.

[0021] In particular, the SRMs methodology may be used as an indicatorof PTGS. As is well known to those skilled in the art, PTGS occurspost-transcriptionally: i.e. the transcription rates of the suppressedgenes are unaffected. The term ‘gene’ is used broadly to describe anysequence which is suitable for translation to a protein.

[0022] Thus the presence of SRMs can be used as a diagnostic test forthe existence of PTGS.

[0023] In one embodiment of this aspect there is disclosed a method ofdetecting or identifying the silencing of a target gene in an organism,which method further comprises characterising any SRMs which arepresent. It should be noted that PTGS effects are very dominant. Inprinciple the presence of SRMs may indicate the silencing of more thanone gene, providing that they have sufficient homology.

[0024] ‘Characterised’ and ‘characterising’ does not necessarily implycomplete sequencing, although this may be preferred. In order to detectsilencing of a known sequence, the SRMs may be fully or partiallysequenced, or sequence identity or similarity may be inferred from e.g.blotting.

[0025] Applications for such a diagnostic test will depend on theorganism in question. For instance, in plants, since PTGS is the basisfor a lot of pathogen derived resistance (PDR), GM field crops (e.g.individuals, or populations) engineered for PDR could be monitored “infield” by checking for the existence of 25 nt RNA to make sure that thePDR was still operating prior to the attack by the virus.

[0026] Similarly, crops depending upon co-suppression for the knockoutof a particular plant gene to achieve a specific modified trait could beassayed for the continued function of PTGS by checking the presence of25 nt RNA against the intended target. Such an assay may be particularlyuseful in view of evidence that transgenes have a tendency to becometranscriptionally inactivated over the generations. PTGS depends upontranscription of the initiating transgene to function and so if thisgets reduced the PTGS will begin to fail. Monitoring 25 nt RNA providesa quick way to test the lines. Non-limiting Examples of silenced geneswhich could be monitored in this way include any of those which havealready been shown to be suppressible by PTGS in the literature. Thesemay include, for example, chalcone synthase of petunia orpolygalacturonase of tomato (Jorgensen, R. A. (1995), Science, 268:686-691, Hamilton, A. J., et al (1995), Current Topics In Microbiologyand Immunology, 197: 77-89).

[0027] It is also possible that the process of PTGS underlies certainplant developmental processes. If there are plant genes which are beingtargeted naturally as a result of PTGS in order to satisfy some plantdevelopmental programme, a 25 nt RNA corresponding to sequences fromthese genes may be detectable.

[0028] Thus, in this embodiment, the SRMs may be used to identify andisolate an unknown target. This could be achieved by analysing the 25nucleotide fraction of RNA from a plant, tagging it with a marker (e.g.a radioactive one) and then using this radioactive RNA to probe alibrary of plant genes. This probe will anneal to genes which areundergoing PTGS in the plant, which genes can then be further analysedor characterised if required. Such genes, inasmuch as they are novel,represent a further aspect of the present invention.

[0029] In a further aspect of the present invention, there is discloseda process for producing or isolating short RNA molecules. As discussedabove, SRMs may not be readily detected by routine RNA analyses,particularly those which include a step in which such molecules are‘lost’ (for instance SRMS may not be efficiently retained on silicacolumns which are used to isolate longer molecules such as mRNAs). Apreferred process is set out in the Examples hereinafter. Broadlyspeaking, the processes provided divide into two parts:extraction/purification and detection.

[0030] For extraction, initial steps may be performed using conventionalRNA extraction methods and kits appropriate to the organism in question,modified as required to ensure that SRMs are retained at each step.

[0031] In order to enhance purification of short RNAs, the extractionmay optionally be followed by one or more of the following steps:

[0032] (i) filtration (e.g. through Centricon 100 concentrators (Amicon)or similar),

[0033] (ii) differential precipitation (e.g. with 5% polyethyleneglycol(8000)/0.5M NaCl)

[0034] (iii) ion exchange chromatography (e.g. using Qiagen columns).

[0035] These steps enrich and purify the short RNAs to greater degreesthan is obtained with the routine rRNA extraction method alone, and maybe performed in conventional manner using, if required, proprietaryreagents.

[0036] It should be noted that there is no requirement that the shortRNAs be purified to homogeneity, provided only that they are capable ofdetection.

[0037] Regarding detection, because of their small size the method forthis is not the usual one for “RNA gel blot analysis” although theprinciple is the same i.e. separation of the RNA molecules according tosize by electrophoresis through a gel.

[0038] Preferably the gel is a 15% polyacrylamide gel containing 7M ureaas a denaturant and TBE (0.5×) as a buffer. The RNAs are preferablytransferred to a hybridisation membrane by electrophoresis (rather thanthe more conventional capillary blot). Once the RNA is on the membrane,it is covalently attached to it by UV irradiation. The membrane is thenplaced in “prehybridisation solution” for a short time.

[0039] Radioactive probe may be prepared using standard techniques.However, preferably, it is made as a single stranded RNA moleculetranscribed in vitro from an appropriate plasmid DNA templates. Thelength of the probe may, preferably, be shortened by limited hydrolysisbefore adding to the prehybridisation solution; this may reducenon-sequence specific binding of probe to the membrane.

[0040] The hybridisation of the probe to its target is allowed toproceed at a stringency level (specific temperature, salt concentrationand the concentration of formamide in the prehybridisation solution)appropriate to the requirements of the process. For instance lowtemperature, high salt, no formamide equals a low stringency, which maypermit short probes or probes with imperfect homology to the target tohybridise with the target. Conversely high temperature, low salt andformamide mean high stringency with only lengthy duplexes stable underthese conditions. Preferred conditions are 45% formamide, 7% SDS, 0.3MNaCl, 0.05M Na₂HPO₄/NaH₂PO₄ (pH7), 1×Denhardt's solution, and singlestranded heterologous nucleic acid (e.g. derived from salmon sperm).

[0041] This is one preferred process of purifying (or partiallypurifying) SRMs for the purpose of detection and/or furthercharacterising e.g. for sequencing. However it should be understood thatthe present invention is in no way limited to this particular format,and others methods for SRMs analysis, such as those which may be devisedin the future, will also be encompassed.

[0042] The process described above may form part of a more extensiveprocess for producing or isolating a silencing agent for a target gene,which silencing agent is a preferably a SRM, the process comprising thesteps of:

[0043] (i) silencing a target gene in an organism,

[0044] (ii) performing a process as described above in order to isolatea SRM appropriate for that gene.

[0045] ‘Silencing agent’ in this context may be one or more of aninducer, signal, or specificity determinant of gene silencing,particularly PTGS. Preferably this will be a SARM (as opposed to aSSRM). Isolated silencing agents obtained or obtainable by this method,inasmuch as they are novel, form a further aspect of the presentinvention.

[0046] The initial silencing step may be achieved by any conventionalmethod appropriate to the organism in question. For instance in plantsit could be by silencing of endogenous, homologous genes(co-suppression—see, for example, van der Krol et al., (1990) The PlantCell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhanget al., (1992) The Plant Cell 4, 1575-1588, and U.S. Pat. No.5,231,020). Further refinements of the gene silencing or co-suppressiontechnology may be found in WO95/34668 (Biosource); Angell & Baulcombe(1997) The EMBO Journal 16, 12:3675-3684; and Voinnet & Baulcombe (1997)Nature 389: pg 553 (systemically induced transgene silencing). Otheroptions include transgene silencing; RNA mediated defence against viralinfection, and transgenic, homology-dependent, virus resistance, or useof dsRNA in the case of nematodes.

[0047] In a further aspect of the present invention there is disclosed amethod for identifying or selecting a target region of a gene, whichgene it is desired to silence, which method comprises:

[0048] (i) silencing the target gene in an organism,

[0049] (ii) performing a process as described above in order to isolatea SRM appropriate for that gene,

[0050] (iii) identifying a region in the sequence of the gene whichcorresponds to the sequence of the SRM.

[0051] The region may identified most readily by comparing the sequenceof the SRM with the sequence of the gene; however any appropriate methodmay be used (e.g. RNAase protection). If several SRMs are isolated, thenseveral target regions may be identified.

[0052] As described in the introduction, this method provides analternative to e.g. computational analysis in order to identify the mostsuitable site on e.g. an mRNA corresponding to a target gene, fortargeting for silencing e.g. with an anti-sense construct. With theinformation obtained using the methods and processes herein about, moreefficient antisense reagents (not necessarily RNAs) may be producedwhich are tailored such that they would be recognised and used by thePTGS machinery of the organism.

[0053] In a further aspect of the present invention there is disclosed amethod of silencing a target gene in an organism which utilises themethodology described above.

[0054] “Silencing” in this context is a term generally used to refer tosuppression of expression of a gene. The degree of reduction may be soas to totally abolish production of the encoded gene product, but moreusually the abolition of expression is partial, with some degree ofexpression remaining. The term should not therefore be taken to requirecomplete “silencing” of expression. It is used herein where convenientbecause those skilled in the art well understand this.

[0055] In one embodiment, the method comprises introducing anti-sensemolecules [SARMs] appropriate for the target gene into the organism inorder to induce silencing. This could be done, for instance, by use oftranscribable constructs encoding the SARMs.

[0056] In a related embodiment, the silencing may be achieved usingconstructs targeting those regions identified by the SRMs-based methoddisclosed above. Such constructs may e.g. encode anti-senseoligonucleotides which target all are part of the identified region, ora region within 1,2,3,4,5,10, 15 or 20 nucleotides of the identifiedregion.

[0057] Suitable target genes for silencing will occur to those skilledin the art as appropriate to the problem in hand. For instance, inplants, it may be desirable to silence genes conferring unwanted traitsin the plant by transformation with transgene constructs containingelements of these genes. Examples of this type of application includesilencing of ripening specific genes in tomato to improve processing andhandling characteristics of the harvested fruit; silencing of genesinvolved in pollen formation so that breeders can reproducibly generatemale sterile plants for the production of F1 hybrids; silencing of genesinvolved in lignin biosynthesis to facilitate paper making fromvegetative tissue of the plant; silencing of genes involved in flowerpigment production to produce novel flower colours; silencing of genesinvolved in regulatory pathways controlling development or environmentalresponses to produce plants with novel growth habit or (for example)disease resistance; elimination of toxic secondary metabolites bysilencing of genes required for toxin production. In addition, silencingcan be useful as a means of developing virus resistant plants when thetransgene is similar to a viral genome.

[0058] As described above, the disclosure herein provides evidence thatSRMs may be a common mediator of PTGS in both plants and higherorganisms. These new findings can be utilised, inter alia, in that itnow appears that induction of SRMs (particularly SARMs) with anappropriate specificity in one organism (say, a plant) may be used tosilence an appropriate target gene in another organism (say, a predator)which comes into contact with that plant.

[0059] In one aspect of the invention there is provided a method fortargeting a gene in a first organism, which method comprises generatinga SARMs silencing agent in a second organism, and introducing the SARMsinto the first organism.

[0060] The SARMs may be generated by any appropriate silencing method.Preferably the target gene will be one which is not an endogenous genein the second organism (but preferably is endogenous to the first). The‘contact’ may be ingestion, injection, or any other method ofadministration. How, precisely, the method is performed will depend onthe organisms and genes involved.

[0061] For instance, in the case of plants and plant predators, it isknown that the systemic signal of PTGS travels out of plant cells intothe phloem (sap) of plants and induces silencing in previouslynon-silencing parts of the plant. In the light of the present disclosureit is clear that, since plant parasitic nematodes feed directly upon thesap and contents of plant cells, they will ingest the signal and inducerof PTGS (i.e. SARMs) in the plant.

[0062] As shown in the Examples below, it appears that the same type ofSARMs are present in C. elegans which are undergoing PTGS induced by theingestion of dsRNA. This implies that the mechanism of PTGS in plantsand nematode is similar if not identical. Thus plant SARMs may triggerthe PTGS of any similar sequences present in the worm. Therefore whenthe nematode feeds on the plant, and eats the PTGS signal, if there ishomology between the plant's transgene from which the PTGS signalderived and a nematode gene, PTGS of that gene ought to be triggered inthe worm.

[0063] Where the targeted gene is an essential gene, this methodprovides a means of controlling or killing plant predators or pests.Naturally, more than one gene can be targeted at once.

[0064] It may be desirable that the targeted gene is one which is eithernot present, or not important, in the wild-type plant or other potentialconsumers of the plant i.e. is nematode specific gene, such as anematode protease gene. This gives the method a high degree ofspecificity.

[0065] Interestingly C. elegans is a nematode distantly related to thenematodes that parasitise plants. Since dsRNA induced PTGS is conservedbetween nematodes, protozoa and insects it is likely that these otherorganisms which support PTGS may be susceptible to SARMs.

[0066] DsRNA interference has also been shown to work in insects andtransgene induced PTGS works in fungi, so it is likely that this is amechanism that is broadly conserved across the kingdoms. This impliesthat any organism that directly feeds off plant cellular contents orextracellular components such as sap could ingest PTGS specific SARMs.If these have sequence homology with genes resident in the parasite,PTGS of these genes could be initiated.

[0067] Thus insect specific genes (e.g. from aphids) represent a furthertarget. Most preferable would be those insect genes or sequences notfound in beneficial insects, such a ladybirds.

[0068] Other targets include genes specific for plant parasites ofplants which feed off the host plant.

[0069] Specifically regarding higher animals (e.g. mammals, fish, birds,reptiles etc.) methods of the present invention include, inter alia:

[0070] (i) methods for detecting or diagnosing gene silencing, orsilencing of particular genes, in the animal by using SRMs as describedabove,

[0071] (ii) methods for identifying silenced genes in the animal byusing SRMs as described above,

[0072] (iii) methods for selecting target sites on genes to be silencedusing SRMs as described above,

[0073] (iv) method for silencing a target gene in the animal, eitherdirectly, or through an animal-derived transgene in a second organism(e.g. a plant) as described above.

[0074] Generally speaking target genes in animals may be those whosefunctional impairment beings therapeutic benefits. Typical genes ofinterest may be (for instance) those involved apoptosis, cancer,cell-cycle regulation, neurological processes, signal transduction etc.Examples and references can be found in the Oncogene Research Products1999 General Catalog, pp 21-265, available from Oncogene ResearchProducts, 84 Rogers Street, Cambridge, Mass. 02142, U.S. Preferredexamples include oncogenes, transcriptional regulators, pocket proteins,members of the MHC superfamily (to produce allotypic organs) etc.

[0075] Some further aspects and applications for the present inventionwill now be discussed.

[0076] According to one aspect of the present invention there isprovided, preferably within a vector suitable for stable transformationof a plant cell, a DNA construct in which a promoter is operably linkedto DNA for transcription in a plant cell to generate either:

[0077] (i) a SARM as described above, or

[0078] (ii) an anti-sense RNA molecule selected to target a regionidentified by the SRM-based methods discussed above.

[0079] Generally speaking, such constructs may be used to silence geneswithin plants, or within organisms predating or being administeredmaterial from plants, in the terms discussed above.

[0080] Anti-sense partial gene sequences selected in accordance withSRM-based methods may be used analogously to those previously used inthe art. See, for example, Rothstein et al, 1987; Smith et al, (1988)Nature 334, 724-726; Zhang et al, (1992) The Plant Cell 4, 1575-1588,English et al., (1996) The Plant Cell 8, 179-188. Antisense technologyis also reviewed in Bourque, (1995), Plant Science 105, 125-149, andFlavell, (1994) PNAS USA 91, 3490-3496. Generally the selected sequencewill be less than 50, 40, 30, 25, or 20 nucleotides. It may bepreferable that there is complete sequence identity in the targeting(e.g. foreign) sequence in the construct and the target sequence in theplant, although total complementarity or similarity of sequence is notessential.

[0081] Again, generally speaking, plants and associated methods andprocesses which form a part of the present invention are either thosewhich:

[0082] (i) are transformed with the ‘targeting’ anti-sense vectors suchas those described above, for instance so as to silence an (endogenous)target gene in the plant or perhaps a viral gene, or

[0083] (ii) are transformed with transgenes taken from other organismssuch as to induce transgene silencing and thereby generate SARMs whichcan be used to silence a target gene in that other organism, or

[0084] (iii) are transformed with vectors which encode SARMs directly,which can be used for either purpose.

[0085] The general methodology discussed below will be applicable to allof these applications.

[0086] A vector which contains the construct may be used intransformation of one or more plant cells to introduce the constructstably into the genome, so that it is stably inherited from onegeneration to the next. This is preferably followed by regeneration of aplant from such cells to produce a transgenic plant. Thus, in furtheraspects, the present invention also provides the use of the construct orvector in production of a transgenic plant, methods of transformation ofcells and plants, plant and microbial (particularly Agrobacterium)cells, and various plant products.

[0087] The function of the promoter in the construct is to ensure thatthe DNA is transcribed into RNA containing the viral sequences. By“promoter” is meant a sequence of nucleotides from which transcriptionmay be initiated of DNA operably linked downstream (i.e. in the 3′direction on the sense strand of double-stranded DNA). A promoter“drives” transcription of an operably linked sequence.

[0088] “Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter.

[0089] Preferred promoters may include the 35S promoter of cauliflowermosaic virus or the nopaline synthase promoter of Agrobacteriumtumefaciens (Sanders, P. R., et al (1987), Nucleic Acids Res., 15:1543-1558). These promoters are expressed in many, if not all, celltypes of many plants. Depending on the target gene of amplicon gs, otherpromoters including those that are developmentally regulated orinducible may be used. For example, if it is necessary to silence thetarget gene specifically in a particular cell type the construct may beassembled with a promoter that drives transcription only in that celltype. Similarly, if the target gene is to be silenced following adefined external stimulus the construct may incorporate a promoter thatis be activated specifically by that stimulus. Promoters that are bothtissue specific and inducible by specific stimuli may be used. Suitablepromoters may include the maize glutathione-S-transferase isoform II(GST-II-27) gene promoter which is activated in response to applicationof exogenous safener (WO93/01294, ICI Ltd).

[0090] An additional optional feature of a construct used in accordancewith the present invention is a transcriptional terminator. Thetranscriptional terminator from nopaline synthase gene of agrobacteriumtumefaciens (Depicker, A., et al (1982), J. Mol. Appl. Genet., 1:561-573) may be used. Other suitable transcriptional terminators will bewell known ot those skilled in the art.

[0091] Those skilled in the art are well able to construct vectors(including those based on ‘naked’ DNA) and design protocols forrecombinant gene expression. For further details see, for example,Molecular Cloning: a Laboratory Manual: 2 nd edition, Sambrook et al,1989, Cold Spring Harbor Laboratory Press. Many known techniques andprotocols for manipulation of nucleic acid, for example in preparationof nucleic acid constructs, mutagenesis, sequencing, introduction of DNAinto cells and gene expression, and analysis of proteins, are describedin detail in Protocols in Molecular Biology, Second Edition, Ausubel etal. eds., John Wiley & Sons, 1992.

[0092] Specific procedures and vectors previously used with wide successupon plants are described by Bevan, Nucl. Acids Res. (1984) 12,8711-8721), and Guerineau and Mullineaux, (1993) Plant transformationand expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed)Oxford, BIOS Scientific Publishers, pp 121-148.

[0093] For introduction into a plant cell, the nucleic acid constructmay be in the form of a recombinant vector, for example an Agrobacteriumbinary vector. Microbial, particularly bacterial and especiallyAgrobacterium, host cells containing a construct according to theinvention or a vector which includes such a construct, particularly abinary vector suitable for stable transformation of a plant cell, arealso provided by the present invention.

[0094] Nucleic acid molecules, constructs and vectors according to thepresent invention may be provided isolated and/or purified (i.e. fromtheir natural environment), in substantially pure or homogeneous form,or free or substantially free of other nucleic acid. Nucleic acidaccording to the present invention may be wholly or partially synthetic.The term “isolate” encompasses all these possibilities.

[0095] An aspect of the present invention is the use of a construct orvector according to the invention in the production of a transgenicplant.

[0096] A further aspect provides a method including introducing theconstruct or vector into a plant cell such that the construct is stablyincorporated into the genome of the cell.

[0097] Any appropriate method of plant transformation may be used togenerate plant cells containing a construct within the genome inaccordance with the present invention. Following transformation, plantsmay be regenerated from transformed plant cells and tissue.

[0098] Successfully transformed cells and/or plants, i.e. with theconstruct incorporated into their genome, may be selected followingintroduction of the nucleic acid into plant cells, optionally followedby regeneration into a plant, e.g. using one or more marker genes suchas antibiotic resistance. Selectable genetic markers may be usedconsisting of chimeric genes that confer selectable phenotypes such asresistance to antibiotics such as kanamycin, hygromycin,phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin,imidazolinones and glyphosate.

[0099] When introducing a nucleic acid into a cell, certainconsiderations must be taken into account, well known to those skilledin the art. The nucleic acid to be inserted should be assembled within aconstruct which contains effective regulatory elements which will drivetranscription. There must be available a method of transporting theconstruct into the cell. Once the construct is within the cell membrane,integration into the endogenous chromosomal material should occur.Finally, as far as plants are concerned the target cell type must besuch that cells can be regenerated into whole plants.

[0100] Plants transformed with the DNA segment containing the sequencemay be produced by standard techniques which are already known for thegenetic manipulation of plants. DNA can be transformed into plant cellsusing any suitable technology, such as a disarmed Ti-plasmid vectorcarried by Agrobacterium exploiting its natural gene transfer ability(EP-A-270355, EP-A-0116718, NAR 12(22) 8711-87215 1984), particle ormicroprojectile bombardment (U.S. Pat. No. 5,100,792, EP-A-444882,EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP175966, Green et al. (1987) Plant Tissue and Cell Culture, AcademicPress), electroporation (EP 290395, WO 8706614 Gelvin Debeyser—

[0101] see attached) other forms of direct DNA uptake (DE 4005152, WO9012096, U.S. Pat. No. 4,684,611), liposome mediated DNA uptake (e.g.Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexingmethod (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d). Physical methods forthe transformation of plant cells are reviewed in Oard, 1991, Biotech.Adv. 9: 1-11.

[0102]Agrobacterium transformation is widely used by those skilled inthe art to transform dicotyledonous species. Recently, there has beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al.(1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al.(1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology9, 957-962; Peng, et al. (1991) International Rice Research Institute,Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11,585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al.(1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990)Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2,603-618; D'Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, etal. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993)Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084;Somers, et al. (1992) Bio/Technology 10, 1589-1594; WO92/14828). Inparticular, Agrobacterium mediated transformation is now emerging alsoas an highly efficient transformation method in monocots (Hiei et al.(1994) The Plant Journal 6, 271-282).

[0103] The generation of fertile transgenic plants has been achieved inthe cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto,K. (1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al.(1992) Bio/Technology 10, 667-674; Vain et al., 1995, BiotechnologyAdvances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page702).

[0104] Microprojectile bombardment, electroporation and direct DNAuptake are preferred where Agrobacterium is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, e.g. bombardmentwith Agrobacterium coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

[0105] Following transformation, a plant may be regenerated, e.g. fromsingle cells, callus tissue or leaf discs, as is standard in the art.Almost any plant can be entirely regenerated from cells, tissues andorgans of the plant. Available techniques are reviewed in Vasil et al.,Cell Culture and Somatic Cel Genetics of Plants, Vol I, II and III,Laboratory Procedures and Their Applications, Academic Press, 1984, andWeissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989.

[0106] The particular choice of a transformation technology will bedetermined by its efficiency to transform certain plant species as wellas the experience and preference of the person practicing the inventionwith a particular methodology of choice. It will be apparent to theskilled person that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

[0107] Also according to the invention there is provided a plant cellhaving incorporated into its genome a DNA construct as disclosed. Afurther aspect of the present invention provides a method of making sucha plant cell involving introduction of a vector including the constructinto a plant cell. Such introduction should be followed by recombinationbetween the vector and the plant cell genome to introduce the sequenceof nucleotides into the genome. RNA encoded by the introduced nucleicacid construct may then be transcribed in the cell and descendantsthereof, including cells in plants regenerated from transformedmaterial. A gene stably incorporated into the genome of a plant ispassed from generation to generation to descendants of the plant, sosuch descendants should show the desired phenotype.

[0108] The present invention also provides a plant comprising a plantcell as disclosed.

[0109] A plant according to the present invention may be one which doesnot breed true in one or more properties. Plant varieties may beexcluded, particularly registrable plant varieties according to PlantBreeders' Rights.

[0110] In addition to a plant, the present invention provides any cloneof such a plant, seed, selfed or hybrid progeny and descendants, and anypart of any of these, such as cuttings, seed.

[0111] The present invention may be used in plants such as crop plants,including cereals and pulses, maize, wheat, potatoes, tapioca, rice,sorgum, millet, cassava, barley, pea and other root, tuber or seedcrops. Important seed crops are oil seed rape, sugar beet, maize,sunflower, soybean and sorghum. Horticultural plants to which thepresent invention may be applied may include lettuce, endive andvegetable brassicas including cabbage, broccoli and cauliflower, andcarnations and geraniums. The present invention may be applied totobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper,chrysanthemum, poplar, eucalyptus and pine.

[0112] In relation to use in mammals or other higher animals, DNAvectors (including naked DNA suitable for expression in mammals) of thepresent invention encode either:

[0113] (i) a SARM as described above, or

[0114] (ii) an anti-sense RNA molecule selected to target a regionidentified by the SRM-based methods discussed above.

[0115] Such vector may be based on any appropriate vector known to thoseskilled in the art. For instance incorporation of this DNA intomammalian cells to produce such antisense RNA in vivo might beaccomplished using vectors based on the disclosure of European patentapplication 909052736.3 (VICAL), HSV, vaccinia or adenovirus (seePrinciples of Gene Manipulation (1994) 5th Edit. Old and Primrose 5thEdition, Blackwell Scientific Publications). Viral vectors for use ingene therapy are discussed by Vile (1997) Nature Biotechnology 15:840-841. A non-viral gene therapy approach is discussed by Sebestyen etal (1998) Nature Biotechnology 16: 80-85. The use of a variety of genetherapy delivery systems (including HSV VP22) is discussed by Fernandez& Baylay (1998) Nature Biotechnology 16: 418-420 and references therein.

[0116] Also provided by the present invention is an organism, preferablya non-human mammal, comprising cells in which a target gene is subjectto PTGS by use of the SARM-based methods or materials disclosed herein.Particularly preferred is a rodent e.g. murine organism. In thisembodiment the invention provides an alternative to known methods ofproducing ‘knock out’ mammals in which specific gene activities havebeen impaired (see e.g. Boerrigter et al (1995) Nature 377: 657-659, orGossen and Vijk (1993) Trends Genet 9: 27-31.)

[0117] The invention will now be further described with reference to thefollowing non-limiting Examples describing work of the inventors. Theresults are also discussed, and suggestions made as to the origin of theSRMs of the present invention. However it will be appreciated by thoseskilled in the art that the materials, methods and processes in thepresent disclosure may be usefully applied irrespective of the preciseunderlying mechanisms involved.

[0118] All references discussed herein, inasmuch as they may be requiredto supplement the present disclosure, are incorporated herein byreference.

EXAMPLES Example 1 Detection of SRMs in Silenced Plants

[0119] Analyses were performed to detect low molecular weight antisenseRNA in four classes of PTGS in plants using the following generalmethods.

[0120] Total RNA was extracted from leaves of tomato, tobacco and N.benthamiana as described previously (E. Mueller, J. E. Gilbert, G.Davenport, G. Brigneti, D. C. Baulcombe, Plant J. 7, 1001 (1995)). Fromthese preparations, low molecular weight RNA was enriched by ionexchange chromatography on Qiagen columns following removal of highmolecular weight species by precipitation with 5% polyethyleneglycol(8000)/0.5M NaCl (for tobacco and N. benthamiana) or (for tomato)by filtration through Centricon 100 concentrators (Amicon). Lowmolecular weight RNA was separated by electrophoresis through 15%polyacrylamide/7M urea/0.5×TBE gels, transferred onto Hybond Nx filters(Amersham) and fixed by UV crosslinking. Prehybridization was in 45%formamide, 7% SDS, 0.3M NaCl, 0.05M Na₂HPO₄/NaH₂PO₄ (pH7), 1×Denhardt'ssolution, 100 mg.ml.⁻¹ sheared, denatured, salmon sperm DNA at between30° C. and 40° C. Hybridization was in the same solution with singlestranded RNA probes transcribed with a-³²P-labelled UTP. Before additionto the filters in the prehybridization solution, probes were hydrolysedto lengths averaging 50 nucleotides. Hybridization was for 16 hours at30° C. (ACO probes), 35° C. (GUS probe) or 40° C. (GFP and PVX probes).

[0121] Sizes of RNA molecules were estimated by comparison with ³³Pphosphorylated DNA oligonucleotides run on the same gels but imagedseparately. Additionally, samples from different types of PTGS includingthose discussed were frequently run on the same gel. Alignment of thefilters following hybridization with different specific probes confirmedthat the PTGS specific signals were identical in size. The probes usedare in each case sequence specific. We have observed nocross-hybridization between 25 nt signals in different PTGS systemsusing either filter hybridisation or RNAase protection

[0122] We do not have an exact measurement of amount of 25 nt per cell,but given the short exposure times routinely used to detect thesemolecules and taking into account their size, they are likely to be veryabundant in cells exhibiting PTGS.

[0123] Co-suppression

[0124] The first class tested was transgene-induced PTGS of anendogenous gene (“co-suppression”). We used five tomato lines (T1.1,T1.2, T5.1, T5.2, T5.3), each transformed with a tomato1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNA sequence placeddownstream of the cauliflower mosaic virus 35S promoter (35S). Two lines(T5.2, T5.3) exhibited PTGS of the endogenous ACO mRNA when amplified byRT-PCR and detected by hybridization with labelled ACO cDNA.

[0125] Low molecular weight nucleic acids purified from the five lineswere separated by denaturing polyacrylamide gel electrophoresis,blotted, and hybridized to an ACO sense (antisense-specific) RNA probe.More specifically, the low molecular weight RNA and a 30-mer ACOantisense RNA oligonucleotide were fractionated, blotted and hybridizedwith either ACO sense RNA or antisense RNA transcribed from full lengthACO cDNA. The low hybridisation temperature permitted some non-specifichybridization to tRNA and small rRNA species which constitute most ofthe RNA mass in these fractions. A discrete, ACO antisense RNA of 25nucleotides (nt) was present in both PTGS lines but absent from thenon-silencing lines. 25 nt ACO RNA of sense polarity and at the sameabundance as the 25 nt ACO antisense RNA was also present only in thePTGS lines. The 25 nt ACO antisense signal was completely abolished bypretreatment with either RNAaseONE or NaOH.

[0126] Transgene Silencing

[0127] PTGS induced by transgenes can also occur when a transgene doesnot have homology to an endogenous gene (1). Therefore we tested whetherthis type of PTGS was also associated with small antisense RNA. Weanalysed three tobacco lines carrying 35S-b-glucuronidase (GUS)transgenes. Two of these lines, T4 (15) and 6b5 (16) exhibited PTGS ofGUS. The third line (6b5×271) tested was produced by crossing 6b5 withline 271 (17) in which there is a transgene suppressor of the35S-promoter in 6b5. There was no PTGS of GUS in 6b5×271 due to thetranscriptional suppression of the 35S GUS transgene (18). Hybridizationwith a GUS-specific probe revealed that low molecular weight GUSantisense RNA was present in T4 and 6b5 but absent from line 6b5×271. 25nt GUS antisense RNA was detected by hybridizhybridisation withhydrolysed GUS sense RNA transcribed from the 3′ 700 bp of the GUS cDNA.The amount of antisense RNA correlated with the degree of PTGS: line 6b5has stronger PTGS of GUS than line T4 (18) and also had more GUSantisense RNA. It appears that 25 nt antisense GUS RNA is dependent upontranscription from the 35S promoter.

[0128] As for PTGS of ACO in tomato, the GUS antisense RNA was adiscrete species of approximately 25 nt.

[0129] Systemically Induced Transgene Silencing

[0130] In some examples of PTGS, silencing is initiated in a localizsedregion of the plant. A signal molecule is produced at the site ofinitiation and mediates systemic spread of silencing to other tissues ofthe plant (19, 20). We investigated whether systemic PTGS of a transgeneencoding the green fluorescent protein (GFP) is associated with 25 ntGFP antisense RNA. PTGS was initiated in Nicotiana benthamianaexpressing a GFP transgene by infiltration of a single leaf withAgrobacterium tumefaciens containing GFP sequences in a binary planttransformation vector.

[0131] More specifically, lower leaves of untransformed N. benthamianaand N. benthamiana carrying an active 35S-GFP transgene (35S-GFP) wereinfiltrated with A. tumefaciens containing the same 35S-GFP transgene ina binary vector. Two to three weeks following this infiltration, the GFPfluorescence disappeared due to systemic spread of PTGS as describedpreviously (11,20).

[0132] RNA from upper, non-infiltrated leaves of these plants and fromequivalent leaves of non-infiltrated plants was hybridized with GFPsense RNA transcribed from a full length GFP cDNA. We detected 25 nt GFPantisense RNA in systemic tissues exhibiting PTGS of GFP. It was notdetected in equivalent leaves of plants that had not been infiltrated orin non-transformed plants that had been infiltrated with the A.tumefaciens i.e. only the transgenic N. benthamiana infiltrated with theA. tumefaciens accumulated 25 nt GFP antisense RNA.

[0133] RNA-mediated Defence Against Viral Infection

[0134] A natural manifestation of PTGS is the RNA-mediated defenceinduced in virus infected cells (8). Therefore we investigated whethervirus-specific, 25 nt RNA could be detected in a virus-infected plant.

[0135] A high titre, synchronised PVX infection on leaves ofuntransformed N. benthamiana. was initiated by infiltration of singleleaves with A. tumefaciens containing a binary plasmid incorporating a35S-PVX-GFP sequence. Once transcribed, the PVX RNA replicon isindependent of the 35S-PVX-GFP DNA, replicates to high levels and movessystemically through the plant. The A. tumefaciens does not spreadbeyond the infiltrated patch and is not present in systemic leaves (20).The GFP reporter in the virus was used to allow visual monitoring ofinfection progress. We have obtained similar signals with wild type PVXinoculated as virions in sap taken from an infected plant.

[0136] RNA was extracted from inoculated leaves after 2, 4, 6 and 10days and from systemic leaves after 6 and 10 days. RNA was extractedfrom mock inoculated leaves after 2 days. 25 nt PVX antisense RNA wasdetected by hybridization with PVX sense RNA transcribed from a fulllength PVX cDNA. 25 nt RNA complementary to the positive strand(genomic) of potato virus X (PVX) was detected 4 days after inoculationof N. benthamiana and continued to accumulate for at least another 8days in the inoculated leaf. 25 nt PVX RNA but was not detected in mockinoculated leaves.

[0137] Discussion

[0138] Thus, 25 nt antisense RNA, complementary to targeted mRNAs,accumulates in four types of PTGS. We have also detected 25 nt RNA inother examples of PTGS as follows: N. benthamiana (spontaneous silencingof a 35S-GFP transgene), tomato (35S-ACO containing an internal directand inverted repeat), petunia (co-suppression of chalcone synthasetransgenes and endogenes) and Arabidopsis thaliana (PTGS of 35S-GFP by a35S-PVX-GFP transgene).

[0139] However the 25 nt RNA has never been detected in the absence ofPTGS. This correlation and the properties of 25 nt RNA are consistentwith a direct role for them in PTGS induced by, for instance, transgenesor viruses (12). 25 nt RNA species also serve as molecular markers forPTGS. Their presence could be used to confirm other examples of e.g.transgene or virus-induced PTGS and may also serve to identifyendogenous genes that are targeted by PTGS in non-transgenic plants. The25 nt antisense RNA species are not degradation products of the targetRNA because they have antisense polarity. A more likely source of theseRNAs is the transcription of an RNA template. This is consistent withthe presence of the 25 nt PVX RNA in PVX infected cells that do notcontain a DNA template. In a further experiment, low molecular weightRNA was extracted from plants containing silencing (S) or non-silencing(NS), 35S-ACC-oxidase (ACO, tomato) or 35S-GFP (N. benthamiana)transgenes. Each was hybridised with ³²P-labelled RNA probes transcribedin the sense orientation from ACC-oxidase and GFP cDNAs and singlestranded RNA then removed by digestion with RNAaseONE (Promega). Theremaining protected RNA molecules were denatured, separated byelectrophoresis on a 15% polyacrylamide/7M urea.0.5×TBE gel. The gel wasdried and imaged by autoradiography. “+” and “−” consist of each probeincubated alone with or without subsequent digestion with RNAaseONE.With the ACO probe, protected fragments are obtained only with RNA fromthe ACO silencing tomato plants and with the GFP probe only with RNAfrom the GFP silencing plants illustrating the sequence specificity ofthe signal. The short RNA species detected in this assay correspond tothe 25 nt RNA detected by northern analysis but are more dispersebecause of RNAase digestion at the ends of breathing RNA duplexes. Somehigher molecular weight signals were also obtained, possible as a resultof incomplete digestion of single stranded regions. The dependency of 25nt GUS antisense RNA accumulation on sense transcription of a GUStransgene also supports the RNA template model. An RNA-dependent RNApolymerase, as required by this model, is required for PTGS inNeurospora crassa (23). With the present data, we cannot distinguishwhether the antisense RNA is made directly as 25 nt species or as longermolecules that are subsequently processed. The precise role of 25 nt RNAin PTGS remains to be determined conclusively. However, as they are longenough to convey sequence specificity yet small enough to move throughplasmodesmata, it is probable that they are components of the systemicsignal and specificity determinants of PTGS.

Example 2 Detection of SRMs in Silenced Nematodes

[0140] RNA from Caenorhabditis elegans was obtained from Department ofEmbryology, Carnegie Institution of Washington, 115 West UniversityParkway, Baltimore, Md. 21210, USA). RNA was extracted by standardmethods known in the art and was concentrated by ethanol precipitationand redissolved in formamide prior to analysis here.

[0141] Nematodes were selected which showed either PTGS (by ingestion ofE. coli which synthesise double stranded GFP RNA) or non-silencing of aGFP transgene.

[0142] Northern analysis of this RNA was performed generally asdescribed above. RNA was fractionated by electrophoresis through a 15%polyacrylamide gel containing 7M urea and 0.5×Tris Borate EDTA bufferand electrophoretically transferred onto a Hybond Nx filter (Amersham)The membrane was placed on three layers of 3MM (Whatman) filter papersaturated with 20×SSC for 20 minutes and then allowed to dry at roomtemperature. The RNA was covalently linked to the membrane byUltraviolet radiation crosslinking (“autocrosslink” setting in“Stratalinker” apparatus (Stratagene). The membrane was prehybridized45% formamide, 7% SDS, 0.3M NaCl, 0.05M Na₂HPO₄/NaH₂PO₄ (pH7),1×Denhardt's solution, 100 mg.ml.⁻¹ sheared, denatured, salmon sperm DNAat 40° C. Hybridization was in the same solution with a single strandedRNA probe transcribed in the sense orientation with α-³²P-labelled UTPfrom the entire coding sequence of GFP. Before addition to the filter inthe prehybridization solution, the probe was hydrolysed to lengthsaveraging approximately 50 nucleotides by incubation in 100 mMNa₂HCO₃/NaH₂CO₃ (pH 10.2) at 60° C. for 3 hours. Hybridization was for16 hours 40° C. The membrane was washed at 50° C. in 2×SSC/0.2% SDS andthe radioactive signal imaged by a phosphorimager.

[0143] As in the previous example, 25 nt. anti-sense RNA was detectablein the silenced material.

References

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1. (Canceled)
 2. A method of silencing a target gene in an organism bypost-transcriptional gene silencing (PTGS), the method comprising thestep of introducing into the organism a silencing agent which targets atargeted region of said target gene, wherein the silencing agentcomprises short RNA molecules (SRMs) which are 25 nucleotides in lengthplus or minus 1, 2, 3, 4 or 5 nucleotides, and which are specific forthe targeted region of the target gene.
 3. The method of claim 2 whereinsaid silencing agent consists of said short RNA molecules.
 4. The methodof claim 2 wherein said silencing agent comprises short RNA moleculeswhich are 21 to 25 nucleotides in length.
 5. The method of claim 2wherein said silencing agent consists of short RNA molecules which are21 to 25 nucleotides in length.
 6. The method of claim 2 wherein saidSRMs are short anti-sense RNA molecules (SARMs) and/or short sense RNAmolecules (SSRMs).
 7. The method of claim 2 wherein said SRMs are shortanti-sense RNA molecules.
 8. The method of claim 2 wherein said SRMs areshort sense RNA molecules.
 9. A method of silencing a target gene in anorganism, comprising (a) providing a DNA construct containing a promoteroperably linked to a DNA which upon transcription in a host cell resultsin a silencing agent specific to a target gene, wherein the silencingagent comprises one or more short RNA molecules (SRMs) which are 25nucleotides in length plus or minus 1, 2, 3, 4 or 5 nucleotides, andwhich silencing agent is specific for a targeted region in a targetgene. (b) introducing said construct into said organism such that thetarget gene in the organism is silenced by the silencing agenttranscribed by said promoter.
 10. The method of claim 9 wherein saidsilencing agent comprises short RNA molecules 21 to 25 nucleotides inlength.
 11. The method of claim 9 wherein said silencing agent comprisesshort RNA molecules (SRMs) wherein said SRMs are short anti-sense RNAmolecules (SARMs) and/or short sense RNA molecules (SSRMs).
 12. Themethod of claim 11 wherein said SRMs are SARMs.
 13. The method of claim11 wherein said SRMs are SSRMs.
 14. A host cell containing a DNAconstruct which comprises a promoter operably linked to DNA which upontranscription in the host cell results in a silencing agent specific toa target gene, and wherein the silencing agent comprises one or moreshort RNA molecules (SRMs) which are 25 nucleotides in length plus orminus 1, 2, 3, 4 or 5 nucleotides, and which SRMs are specific for atargeted region in a target gene and upon transcription silence thetarget gene.
 15. A method of selecting a target region in a target genewhich is desired to be silenced comprising: (I) isolating one or moreRNA molecules from a sample, wherein said RNA molecules are short RNAmolecules (SRMs) which are 25 nucleotides in length plus or minus 1, 2,3, 4 or 5 nucleotides, and which are specific for a target region of atarget gene by: (a) producing a nucleic acid extract from said sample;(b) purifying said extract to obtain purified RNA molecules by effectingat least one purification step selected from the group consisting of (i)filtration; (ii) differential precipitation and (iii) ion exchangechromatography and isolating SRMs which are silencing agents for saidtarget gene; (II) identifying a target region in the sequence of saidtarget gene which corresponds to a sequence comprised in said SRMs. 16.The method of claim 15 which further comprises separating the purifiedRNA molecules according to size by gel electrophoresis using a 15%polyacrylamide gel containing 7M urea as a denaturant and TBE (0.5×) asa buffer.
 17. The method of claim 16 which further comprisestransferring the RNA molecules comprised on the gel to a hybridizationmembrane by electrophoresis.
 18. The method of claim 17 which furthercomprises labeling the RNA molecules comprised on the hybridizationmembrane using a radioactive probe obtained from a single stranded RNAmolecule transcribed in vitro from a plasmid DNA template.
 19. A methodof silencing a target gene in an organism comprising: (i) performing amethod according to claim 15 to select a target region of a target geneto be silenced; and (ii) silencing said target gene in an organism bytargeting said target region with a silencing agent.
 20. The method ofclaim 19 wherein step (ii) is effected by introducing into the organismSRMs specific to the targeted region of the target gene which inducesilencing of said target gene.
 21. The method of claim 20 wherein saidSRMs comprise RNA molecules which are 25 nucleotides in length plus orminus 1, 2, 3, 4 or 5 nucleotides.
 22. The method of claim 21 whereinsaid SRMs comprise RNAs which are 21 to 25 nucleotides in length.
 23. Amethod of silencing a target gene in a first organism comprising: (i)generating in a second organism short RNA molecules (SRMs) which are asilencing agent for said target gene, wherein said SRMs are 25nucleotides in length plus or minus 1, 2, 3, 4 or 5 nucleotides, and arespecific for a target region in said target gene; and (ii) introducingsaid SRMs into said first organism in order to silence said target genecomprised therein.
 24. The method of claim 23 wherein said SRMs are 21to 25 nucleotides in length.
 25. The method of claim 23 wherein saidSRMs are short anti-sense RNA molecules (SARMs) and/or short sense RNAmolecules (SSRMs)
 26. The method of claim 23 wherein said SRMs areSARMs.
 27. The method of claim 23 wherein said SRMs are SSRMs.
 28. Themethod of claim 23 wherein said target gene is endogenous to the firstorganism but is not endogenous to the second organism.